Mutations in the PX-SH3A linker of p47phox decouple PI(3,4)P2 binding from NADPH oxidase activation

NADPH oxidase is essential in the human innate immune response. p47 (phox), a cytosolic NADPH oxidase component, plays a regulatory role in the activation of NADPH oxidase. Our manipulation of p47 (phox) by mutation and amino acid deletion shows that the linker region between the PX and N-terminal SH3 domain plays a role in blocking the binding of the phosphoinositide 3,4-bisphosphate [PI(3,4)P2], a lipid second messenger generated upon neutrophil activation. Replacement of linker residues 151-158 with glycine alters NMR-measured spin lattice relaxation rates and sedimentation velocity compared to those of the wild-type protein, suggesting that the PX domain is released from its autoinhibited conformation. Liposome binding and surface plasmon resonance experiments confirm this result, showing that this mutant has a similar binding affinity for the isolated PX domain toward PI(3,4)P2. However, an in vitro NADPH oxidase activity assay reveals that this glycine mutant of the full-length protein greatly reduced NADPH oxidase activity upon activation even though it displayed PI(3,4)P2 binding activity comparable to that of the isolated PX domain. Our results highlight an active role of the PX-SH3 linker region in maintaining p47 (phox) in its fully autoinhibited form and demonstrate that binding of p47 (phox) to membrane phospholipids is mechanistically distinct from NADPH oxidase activation.     

Membrane binding mechanisms of the PX domains of NADPH oxidase p40phox and p47phox

Phox (PX) domains are phosphoinositide (PI)-binding domains with broad PI specificity. Two cytosolic components of NADPH oxidase, p40(phox) and p47(phox), contain PX domains. The PX domain of p40(phox) specifically binds phosphatidylinositol 3-phosphate, whereas the PX domain of p47(phox) has two lipid binding sites, one specific for phosphatidylinositol 3,4-bisphosphate and the other with affinity for phosphatidic acid or phosphatidylserine. To delineate the mechanisms by which these PX domains interact with PI-containing membranes, we measured the membrane binding of these domains and respective mutants by surface plasmon resonance and monolayer techniques and also calculated the electrostatic potentials of the domains as a function of PI binding. Results indicate that membrane binding of both PX domains is initiated by nonspecific electrostatic interactions, which is followed by the membrane penetration of hydrophobic residues. The membrane penetration of the p40(phox) PX domain is induced by phosphatidylinositol 3-phosphate, whereas that of the p47(phox) PX domain is triggered by both phosphatidylinositol 3,4-bisphosphate and phosphatidic acid (or phosphatidylserine). Studies of enhanced green fluorescent protein-fused PX domains in HEK293 cells indicate that this specific membrane penetration is also important for subcellular localization of the two PX domains. Further studies on the full-length p40(phox) and p47(phox) proteins showed that an intramolecular interaction between the C-terminal Src homology 3 domain and the PX domain prevents the nonspecific monolayer penetration of p47(phox), whereas such an interaction is absent in p40(phox).     

Atypical membrane-embedded phosphatidylinositol 3,4-bisphosphate (PI(3,4)P2)-binding site on p47(phox) Phox homology (PX) domain revealed by NMR

The Phox homology (PX) domain is a functional module that targets membranes through specific interactions with phosphoinositides. The p47(phox) PX domain preferably binds phosphatidylinositol 3,4-bisphosphate (PI(3,4)P(2)) and plays a pivotal role in the assembly of phagocyte NADPH oxidase. We describe the PI(3,4)P(2) binding mode of the p47(phox) PX domain as identified by a transferred cross-saturation experiment. The identified PI(3,4)P(2)-binding site, which includes the residues of helices α1 and α1' and the following loop up to the distorted left-handed PP(II) helix, is located at a unique position, as compared with the phosphoinositide-binding sites of all other PX domains characterized thus far. Mutational analyses corroborated the results of the transferred cross-saturation experiments. Moreover, experiments with intact cells demonstrated the importance of this unique binding site for the function of the NADPH oxidase. The low affinity and selectivity of the atypical phosphoinositide-binding site on the p47(phox) PX domain suggest that different types of phosphoinositides sequentially bind to the p47(phox) PX domain, allowing the regulation of the multiple events that characterize the assembly and activation of phagocyte NADPH oxidase.     

p47(phox) PX domain of NADPH oxidase targets cell membrane via moesin-mediated association with the actin cytoskeleton

Activation of phagocytic NADPH oxidase requires association of its cytosolic subunits with the membrane-bound flavocytochrome. Extensive phosphorylation of the p47(phox) subunit of NADPH oxidase marks the initiation of this activation process. The p47(phox) subunit then translocates to the plasma membrane, bringing the p67(phox) subunit to cytochrome b558 to form the active NADPH oxidase complex. However, the detailed mechanism for targeting the p47(phox) subunit to the cell membrane during activation still remains unclear. Here, we show that the p47(phox) PX domain is responsible for translocating the p47(phox) subunit to the plasma membrane for subsequent activation of NADPH oxidase. We also demonstrate that translocation of the p47(phox) PX domain to the plasma membrane is not due to interactions with phospholipids but rather to association with the actin cytoskeleton. This association is mediated by direct interaction between the p47(phox) PX domain and moesin.     

Full-length p40phox structure suggests a basis for regulation mechanism of its membrane binding

The superoxide-producing phagocyte NADPH oxidase is activated during phagocytosis to destroy ingested microbes. The adaptor protein p40phox associates via the PB1 domain with the essential oxidase activator p67phox, and is considered to function by recruiting p67phox to phagosomes; in this process, the PX domain of p40phox binds to phosphatidylinositol 3-phosphate [PtdIns(3)P], a lipid abundant in the phagosomal membrane. Here we show that the PtdIns(3)P-binding activity of p40phox is normally inhibited by the PB1 domain both in vivo and in vitro. The crystal structure of the full-length p40phox reveals that the inhibition is mediated via intramolecular interaction between the PB1 and PX domains. The interface of the p40phox PB1 domain for the PX domain localizes on the opposite side of that for the p67phox PB1 domain, and thus the PB1-mediated PX regulation occurs without preventing the PB1-PB1 association with p67phox.     

Phosphatidylinositol 3-phosphate-dependent and -independent functions of p40phox in activation of the neutrophil NADPH oxidase

In response to bacterial infection, the neutrophil NADPH oxidase assembles on phagolysosomes to catalyze the transfer of electrons from NADPH to oxygen, forming superoxide and downstream reactive oxygen species (ROS). The active oxidase is composed of a membrane-bound cytochrome together with three cytosolic phox proteins, p40(phox), p47(phox), and p67(phox), and the small GTPase Rac2, and is regulated through a process involving protein kinase C, MAPK, and phosphatidylinositol 3-kinase. The role of p40(phox) remains less well defined than those of p47(phox) and p67(phox). We investigated the biological role of p40(phox) in differentiated PLB-985 neutrophils, and we show that depletion of endogenous p40(phox) using lentiviral short hairpin RNA reduces ROS production and impairs bacterial killing under conditions where p67(phox) levels remain constant. Biochemical studies using a cytosol-reconstituted permeabilized human neutrophil cores system that recapitulates intracellular oxidase activation revealed that depletion of p40(phox) reduces both the maximal rate and total amount of ROS produced without altering the K(M) value of the oxidase for NADPH. Using a series of mutants, p47PX-p40(phox) chimeras, and deletion constructs, we found that the p40(phox) PX domain has phosphatidylinositol 3-phosphate (PtdIns(3)P)-dependent and -independent functions. Translocation of p67(phox) requires the PX domain but not 3-phosphoinositide binding. Activation of the oxidase by p40(phox), however, requires both PtdIns(3)P binding and an Src homology 3 (SH3) domain competent to bind to poly-Pro ligands. Mutations that disrupt the closed auto-inhibited form of full-length p40(phox) can increase oxidase activity approximately 2.5-fold above that of wild-type p40(phox) but maintain the requirement for PX and SH3 domain function. We present a model where p40(phox) translocates p67(phox) to the region of the cytochrome and subsequently switches the oxidase to an activated state dependent upon PtdIns(3)P and SH3 domain engagement.     

NOXO1, regulation of lipid binding, localization, and activation of Nox1 by the Phox homology (PX) domain

NOXO1 (Nox organizing protein 1) and NOXA1 (Nox activating protein 1) are homologs of p47phox and p67phox. p47phox functions in phagocytes as an essential organizing protein mediating the binding of other regulatory proteins during activation of the phagocyte oxidase, and its translocation to the membrane is triggered upon cell activation by hyperphosphorylation, which relieves autoinhibition of SH3 and PX domains. NOXO1 lacks an autoinhibitory region and phosphorylation sites that are present in p47phox. Co-transfection of Nox1, NOXO1, and NOXA1 reconstitutes ROS (reactive oxygen species) generation in HEK293 cells in the absence of cell stimulation. NOXO1 binds to the phosphatidylinositol (PtdIns) lipids PtdIns 3,5-P2, PtdIns 5-P, and PtdIns 4-P. Unlike p47phox, which is located in the cytosol of resting cells and translocates to the plasma membrane where gp91phox is located, NOXO1 co-localizes with Nox1 in the membranes of resting cells. This localization of NOXO1 is dictated by its PX domain, since this domain but not the remainder of the molecule localizes to membranes. A point mutation in the PX domain of holo-NOXO1 decreases lipid binding resulting in cytosolic localization and also inhibits NOXO1-activation of Nox1. Thus, in transfected HEK293 cells, NOXO1 and NOXA1 activate Nox1 without the need for agonist activation, and this is mediated in part by binding of the NOXO1 PX domain to membrane lipids.     

The crystal structure of the PX domain from p40(phox) bound to phosphatidylinositol 3-phosphate.

More than 50 human proteins with a wide range of functions have a 120 residue phosphoinositide binding module known as the PX domain. The 1.7 A X-ray crystal structure of the PX domain from the p40(phox) subunit of NADPH oxidase bound to PtdIns(3)P shows that the PX domain embraces the 3-phosphate on one side of a water-filled, positively charged pocket and reveals how 3-phosphoinositide specificity is achieved. A chronic granulomatous disease (CGD)-associated mutation in the p47(phox) PX domain that abrogates PtdIns(3)P binding maps to a conserved Arg that does not directly interact with the phosphoinositide but instead appears to stabilize a critical lipid binding loop. The SH3 domain present in the full-length protein does not affect soluble PtdIns(3)P binding to the p40(phox) PX domain.     

The p47phox PX domain: two heads are better than one!

The recent X-ray structure of the PX domain of p47phox, a critical subunit of the NADPH oxidase, unexpectedly revealed the presence of two distinct lipid binding pockets within this single modular domain. This unusual feature allows the p47phox PX domain to integrate signal transduction events emerging from two different lipid signaling pathways.     

The role of PI3Ks in the regulation of the neutrophil NADPH oxidase

The NADPH oxidase complex of neutrophils and macrophages is an important weapon used by these cells to kill microbial pathogens. The regulation of this enzyme complex is necessarily complicated by the diverse receptor types that are needed to trigger its activation and also the tight control that is required to deliver this activation at the appropriate time and place. As such, several signalling pathways have been established to regulate the NADPH oxidase downstream of cell surface receptors. Central amongst these are PI3K- (phosphoinositide 3-kinase)-dependent pathways, blockade of which severely limits activation of the oxidase to several soluble and particulate stimuli. The precise roles of the phosphoinositide products of PI3K activity in regulating NADPH oxidase assembly and activation are still unclear, but there is emerging evidence that they play a key role via regulation of guanine nucleotide exchange on Rac, a key component in the oxidase complex. There is also very strong evidence that the PI3K products PtdIns(3,4)P2 and PtdIns3P can bind directly to the PX (Phox homology) domains of the core oxidase components p47phox and p40phox respectively. However, the significance of these interactions in terms of membrane localization or allosteric consequences for the oxidase complex remains to be established.     

Characterization of a mutation in the Phox homology domain of the NADPH oxidase component p40phox identifies a mechanism for negative regulation of superoxide production

The phagocyte oxidase (Phox) protein p40(phox) contains a Phox homology (PX) domain which, when expressed alone, interacts with phosphatidylinositol 3-phosphate (PtdIns (3)P). The functions of the PX domain in p40(phox) localization, association with the cytoskeleton, and superoxide production were examined in transgenic COS-7 cells expressing gp91(phox), p22(phox), p67(phox), and p47(phox) (COS(phox) cells). Full-length p40(phox) exhibited a cytoplasmic localization pattern in resting cells. Upon stimulation with phorbol 12-myristate 13-acetate or fMet-Leu-Phe, p40(phox) translocated to plasma membrane in a p67(phox)- and p47(phox)-dependent manner. Heterologous expression of p40(phox) markedly enhanced superoxide production in phorbol 12-myristate 13-acetate - and fMet-Leu-Phe-stimulated COS(phox) cells. Unexpectedly, mutation of Arg-57 in the PX domain to Gln, which abrogated PtdIns (3)P binding, produced a dominant inhibitory effect on agonist-induced superoxide production and membrane translocation of p47(phox) and p67(phox). The mutant p40(phox) (p40R57Q) displayed increased association with actin and moesin and was found enriched in the Triton X-100-insoluble fraction along with p67(phox) and p47(phox). The enhanced cytoskeleton association of p67(phox) and p47(phox) and the dominant inhibitory effect produced by the p40R57Q were alleviated when a second mutation at Asp-289, which eliminated p40(phox) interaction with p67(phox), was introduced. Likewise, cytochalasin B treatment abolished the dominant inhibitory effect of p40R57Q on superoxide production. These findings suggest a dual regulatory mechanism through the PX domain of p40(phox); its interaction with the actin cytoskeleton may stabilize NADPH oxidase in resting cells, and its binding of PtdIns (3)P potentiates superoxide production upon agonist stimulation. Both functions require the association of p40(phox) with p67(phox).     

A new role of Pro-73 of p47phox in the activation of neutrophil NADPH oxidase

The PX domain of p47phox is thought to be involved in autoinhibition. However, when the domain was deleted, the ability to activate the phagocyte NADPH oxidase was markedly diminished. We have mutated the proline-rich region of the PX domain and examined the mutants for the ability to activate. Substitution of Gln for Pro-73 of p47phox(1-286) (P73Q) resulted in a considerably lower activity than the wild type and P73Q had a much lower affinity for the oxidase complex. Whereas, Gln substitution for Pro-76 (P76Q) showed a slightly enhanced activation and the mutant had a slightly higher affinity for the complex than the wild type. Affinity for p67phox(1-210) was slightly decreased either by P73Q or P76Q. Optimal SDS concentration for the activation was lowered by these mutations. Binding of PX domain with phosphatidylinositol-3,4-bisphosphate was diminished by P73Q mutation. The results in this study suggest that Pro-73 has a role in interaction with the catalytic component cytochrome b558.     

Tripartite chimeras comprising functional domains derived from the cytosolic NADPH oxidase components p47phox, p67phox, and Rac1 elicit activator-independent superoxide production by phagocyte membranes: an essential role for anionic membrane phospholipids

The superoxide-generating NADPH oxidase is converted to an active state by the assembly of a membrane-localized cytochrome b(559) with three cytosolic components: p47(phox), p67(phox), and GTPase Rac1 or Rac2. Assembly involves two sets of protein-protein interactions: among cytosolic components and among cytosolic components and cytochrome b(559) within its lipid habitat. We circumvented the need for interactions among cytosolic components by constructing a recombinant tripartite chimera (trimera) consisting of the Phox homology (PX) and Src homology 3 (SH3) domains of p47(phox), the tetratricopeptide repeat and activation domains of p67(phox), and full-length Rac1. Upon addition to phagocyte membrane, the trimera was capable of oxidase activation in vitro in the presence of an anionic amphiphile. The trimera had a higher affinity (lower EC(50)) for and formed a more stable complex (longer half-life) with cytochrome b(559) compared with the combined individual components, full-length or truncated. Supplementation of membrane with anionic but not neutral phospholipids made activation by the trimera amphiphile-independent. Mutagenesis, truncations, and domain replacements revealed that oxidase activation by the trimera was dependent on the following interactions: 1) interaction with anionic membrane phospholipids via the poly-basic stretch at the C terminus of the Rac1 segment; 2) interaction with p22(phox) via Trp(193) in the N-terminal SH3 domain of the p47(phox) segment, supplementing the electrostatic attraction; and 3) an intrachimeric bond among the p67(phox) and Rac1 segments complementary to their physical fusion. The PX domain of the p47(phox) segment and the insert domain of the Rac1 segment made only minor contributions to oxidase assembly.     

The PX domain as a novel phosphoinositide- binding module

The phox (phagocyte oxidase) homology (PX) domain occurs in the mammalian phox proteins p40(phox) and p47(phox), the polarity establishment protein Bem1p in budding yeast, and a variety of proteins involved in membrane trafficking. Here we show that the PX domains of p40(phox) and p47(phox) directly bind to phosphoinositides: p40(phox) prefers Ptdlns(3)P, while p47(phox) does Ptdlns(4)P and Ptdlns(3,4)P(2). In addition, the Bem1p PX domain also interacts with Ptdlns(4)P. When the p40(phox) PX domain is expressed as a fusion to green fluorescent protein in HeLa cells, it exists at early endosomes where Ptdlns(3)P is enriched. Furthermore, a mutant p40(phox) PX carrying the substitution of Lys for Arg105 only weakly binds to phosphoinositides in vitro, and fails to locate to early endosomes. Thus the PX domain functions as a novel phosphoinositide-binding module and likely participates in targeting of proteins to membranes.     

Effects of p47phox C terminus phosphorylations on binding interactions with p40phox and p67phox. Structural and functional comparison of p40phox and p67phox SH3 domains

The neutrophil NADPH oxidase produces superoxide anions in response to infection. This reaction is activated by association of cytosolic factors, p47phox and p67phox, and a small G protein Rac with the membranous flavocytochrome b558. Another cytosolic factor, p40phox, is associated to the complex and is reported to play regulatory roles. Initiation of the NADPH oxidase activation cascade has been reported as consecutive to phosphorylation on serines 359/370 and 379 of the p47phox C terminus. These serines surround a polyproline motif that can interact with the Src homology 3 (SH3) module of p40phox (SH3p40) or the C-terminal SH3 of p67phox (C-SH3p67). The latter one presents a higher affinity in the resting state for p47phox. A change in SH3 binding preference following phosphorylation has been postulated earlier. Here we report the crystal structures of SH3p40 alone or in complex with a 12-residue proline-rich region of p47phox at 1.46 angstrom resolution. Using intrinsic tryptophan fluorescence measurements, we compared the affinity of the strict polyproline motif and the whole C terminus peptide with both SH3p40 and C-SH3p67. These data reveal that SH3p40 can interact with a consensus polyproline motif but also with a noncanonical motif of the p47phox C terminus. The electrostatic surfaces of both SH3 are very different, and therefore the binding preference for C-SH3p67 can be attributed to the polyproline motif recognition and particularly to the Arg-368p47 binding mode. The noncanonical motif contributes equally to interaction with both SH3. The influence of serine phosphorylation on residues 359/370 and 379 on the affinity for both SH3 domains has been checked. We conclude that contrarily to previous suggestions, phosphorylation of Ser-359/370 does not modify the SH3 binding affinity for both SH3, whereas phosphorylation of Ser-379 has a destabilizing effect on both interactions. Other mechanisms than a phosphorylation induced switch between the two SH3 must therefore take place for NADPH oxidase activation cascade to start.     

Solution structure of the PX domain, a target of the SH3 domain

The phox homology (PX) domain is a novel protein module containing a conserved proline-rich motif. We have shown that the PX domain isolated from the human p47phox protein, a soluble subunit of phagocyte NADPH oxidase, binds specifically to the C-terminal SH3 domain derived from the same protein. The solution structure of p47 PX has an alpha + beta structure with a novel folding motif topology and reveals that the proline-rich motif is presented on the molecular surface for easy recognition by the SH3 domain. The proline-rich motif of p47 PX in the free state adopts a distorted left-handed polyproline type II helix conformation.     

The p40phox and p47phox PX domains of NADPH oxidase target cell membranes via direct and indirect recruitment by phosphoinositides.

The Phox homology (PX) domain has recently been reported to bind to phosphoinositides, and some PX domains can localize to endosomes in vivo. Here we show data to support the conclusion that the p40(phox) PX domain binds to phosphatidylinositol 3-phosphate specifically in vitro and localizes to endosomes in intact cells. In addition, its Y59A/L65Q mutant, which has decreased affinity for phosphatidylinositol 3-phosphate in vitro, fails to target EGFP-p40-PX to endosomes. However, unlike published results, we find that the p47(phox) PX domain weakly binds to many phosphoinositides in vitro showing slightly higher affinity for phosphatidylinositol 3,4,5-trisphosphate. Moreover, we show for the first time that upon insulin-like growth factor-1 stimulation of COS cells, the p47(phox) PX domain is localized to the plasma membrane, and this subcellular localization is dependent on PI 3-kinase activity. Unexpectedly, its R42Q mutant that loses in vitro phosphoinositide-binding ability can still target EGFP-p47-PX to the plasma membrane. Our data suggest that the translocation of p47(phox) PX domain to the plasma membrane does involve 3'-phosphoinositide(s) in the process, but the phosphoinositide-binding of p47(phox) PX domain is not sufficient to recruit it to the plasma membrane. Therefore, the p40(phox) and p47(phox) PX domains can target subcellular membranes via direct or indirect recruitment by phosphoinositides, while both are under the control of phosphatidylinositol 3-kinase activity.     

Solution structure of the Vam7p PX domain

PX domains have been recently found to act as phosphoinositide binding modules. In the yeast SNARE protein Vam7p, the PX domain binds to PtdIns(3)P and is required for vacuolar targeting. To gain insight into how PX domains function, the solution structure of the ligand-free Vam7p PX domain has been determined by NMR spectroscopy. The Vam7p PX domain has the same overall alpha/beta fold observed in the structures of the ligand-free p47(phox) PX domain and the PtdIns(3)P-bound p40(phox) PX domain, exhibiting several similarities and differences with these two PX domains. Most striking is the similarity between the Vam7p and p40(phox) PX domains in a subset of secondary structure elements despite the low level of sequence identity between them, suggesting that these elements form a conserved core in the PX domain fold. These similarities and the observation that a putative PtdIns(3)P binding site is already formed in the apo Vam7p PX domains suggest that ligand binding does not induce major conformational changes, contrary to what was previously thought. The proposed ligand binding site of the Vam7p PX domain includes basic side chains from the conserved structural core that also participate in PtdIns(3)P binding to the p40(phox) PX domain, and basic side chains from a variable loop that probably inserts into the membrane. These results indicate that PX domains contain a combination of conserved and variable features that allow them to have a common function and at the same time exhibit distinct specificities, mechanisms of regulation, or modes of interaction with effector molecules.     

PtdIns3P binding to the PX domain of p40phox is a physiological signal in NADPH oxidase activation

The production of reactive oxygen species by the NADPH oxidase complex of phagocytes plays a critical role in our defence against bacterial and fungal infections. The PX domains of two oxidase components, p47(phox) and p40(phox), are known to bind phosphoinositide products of PI3Ks but the physiological roles of these interactions are unclear. We have created mice which carry an R58A mutation in the PX domain of their p40(phox) gene, which selectively prevents binding to PtdIns3P. p40(phoxR58A/R58A) embryos do not develop normally but p40(phoxR58A/-) mice are viable and neutrophils from these animals exhibit significantly reduced oxidase responses compared to those from their p40(phox+/-) siblings (e.g. 60% reduced in response to phagocytosis of Staphylococcus aureus). Wortmannin inhibition of the S. aureus oxidase response correlates with inhibition of phagosomal PtdIns3P accumulation and overlaps with the reduction in this response caused by the R58A mutation, suggesting PI3K regulation of this response is substantially dependent on PtdIns3P-binding to p40(phox). p40(phoxR58A/-) mice are significantly compromised in their ability to kill S. aureus in vivo, defining the physiological importance of this interaction.     

Mapping of functional domains in the p22(phox) subunit of flavocytochrome b(559) participating in the assembly of the NADPH oxidase complex by "peptide walking".

The superoxide-generating NADPH oxidase complex of phagocytes consists of a membranal heterodimeric flavocytochrome (cytochrome b(559)), composed of gp91(phox) and p22(phox) subunits, and four cytosolic proteins, p47(phox), p67(phox), p40(phox), and the small GTPase Rac (1 or 2). All redox stations involved in electron transport from NADPH to oxygen are located in gp91(phox). NADPH oxidase activation is the consequence of assembly of cytochrome b(559) with cytosolic proteins, a process reproducible in a cell-free system, consisting of phagocyte membranes, and recombinant cytosolic components, activated by an anionic amphiphile. p22(phox) is believed to act as a linker between the cytosolic components and gp91(phox). We applied "peptide walking" to mapping of domains in p22(phox) participating in NADPH oxidase assembly. Ninety one synthetic overlapping pentadecapeptides, spanning the p22(phox) sequence, were tested for the ability to inhibit NADPH oxidase activation in the cell-free system and to bind individual cytosolic NADPH oxidase components. We conclude the following. 1) The p22(phox) subunit of cytochrome b(559) serves as an anchor for both p47(phox) and p67(phox). 2) p47(phox) binds not only to the proline-rich region, located at residues 151-160 in the cytosolic C terminus of p22(phox), but also to a domain (residues 51-63) located on a loop exposed to the cytosol. 3) p67(phox) shares with p47(phox) the ability to bind to the proline-rich region (residues 151-160) and also binds to two additional domains, in the cytosolic loop (residues 81-91) and at the start of the cytosolic tail (residues 111-115). 4) The binding affinity of p67(phox) for p22(phox) peptides is lower than that of p47(phox). 5) Binding of both p47(phox) and p67(phox) to proline-rich p22(phox) peptides occurs in the absence of an anionic amphiphile. A revised membrane topology model of p22(phox) is proposed, the core of which is the presence of a functionally important cytosolic loop (residues 51-91).